Labeling Of Phosphatidylinositol Research Articles

Formation and remodeling of inositolphosphoceramide during differentiation of Trypanosoma cruzi from trypomastigote to amastigote.

Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [(3)H]palmitic acid or [(3)H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using (3)H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A(1), phospholipase A(2), inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

Myo-[ 3H]-inositol loaded erythrocytes and white ghosts: Two models to investigate the phosphatidylinositol synthesis in human red cells

Human erythrocytes were loaded with myo-[ 3H]-inositol in the presence or absence of cytidine trisphosphate to investigate the synthesis of membrane phosphoinositides in the intact red cell. The addition of cytidylic nucleotides to the loading mixture yielded a four-fold increase in the [ 3H]-labeling of the membranes. The [ 3H]-labeling of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was distinguished by two chromatographic techniques. Experiments performed on white ghosts demonstrated the presence of CDP-diacylglycerol synthase and phosphatidylinositol synthase. These results and those already reported allow to discuss a possible turnover of the inositol polar head.

Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland

The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4–9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2) which reflects PI cycle activation. AVT (10 −5 and 10 −4 M) induced a significant increase in 32P labelling of PI, PIP and PIP 2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V 1 receptor antagonist. The desmopressin and AVP 4–9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 μg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

Diacylglycerols derived from membrane phospholipids are metabolized by lipases in A10 smooth muscle cells.

The metabolic fate of endogenous diacylglycerol (DAG) in cultured A10 smooth muscle cells was determined. Preincubation of A10 cells with [3H]myristic acid or [3H]arachidonic acid resulted in preferential labeling of phosphatidylcholine (PC) or phosphatidylinositol (PI), respectively. Addition of PC-specific phospholipase C (PC-PLC) to [3H]myristate-labeled A10 cells resulted in a 10-fold increase in radiolabeled DAG, which was converted to monoacylglycerol (MG) and fatty acid (FA). DAG degradation and MG formation was inhibited by tetrahydrolipstatin, a DAG lipase inhibitor. PC-derived DAG was not converted to phosphatidic acid; in addition, PC resynthesis or triacylglycerol synthesis was not observed. Addition of PI-specific PLC (PI-PLC) to [3H]arachidonate-labeled A10 cells resulted in a modest increase in radiolabeled DAG that was also hydrolyzed to MG and FA. Therefore, the principal metabolic fate of endogenous DAG generated from membrane phospholipids by treatment of A10 cells with PC-PLC and PI-PLC was hydrolysis by a DAG lipase pathway.

Arginine vasopressin stimulates 32P labeling of phosphoinositides in rat pineal gland

The peptidergic innervation of the pineal gland contains arginine vasopressin (AVP) fibers. Since the site and molecular mechanism of AVP action on the pineal signal transduction has not yet been determined, we examined whether the inositol phosphate transduction system is involved. The phosphoinositide signaling system was studied by measuring metabolic turnover of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol biphosphate (PIP 2), which reflects phosphoinositide cycle activation. AVP induced a significant increase in 32P labeling of PI, PIP and PIP 2. The V 1 receptor site(s) blocker for AVP abolished enhanced 32P labeling of PIP and PIP 2. AVP was without effect on cAMP dependent phosphorylation. The data indicate that AVP activates the phosphoinositide signaling system via V 1 receptors and is without effect on the cAMP system.

Regulation of the Activity and Phosphorylation of the Plasma Membrane Ca 2+-ATPase by Adriamycin in Intact Human Erythrocytes

We have previously shown in intact human erythrocytes that both the plasma membrane Ca 2+ pump activity and its phosphorylation can be increased by phorbol-la-myristate 13-acetate (PMA), a known stimulator of protein kinase C. These effects were inhibited by high doses of adriamycin (L. C. Wright et al., 1993, Arch. Biochem. Biophys. 306, 277-284). We now show that low doses of adriamycin (ADR) (maximum effect at 10 μM for 1-6 min) decrease the amplitude of the intracellular calcium ([Ca 2+] i) transient induced by 2.5 μM CaCl 2 and 10 μM A23187 in intact human erythrocytes, This is reflected by a parallel increase in Ca 2+-ATPase activity in plasma membranes isolated from pretreated intact cells. When 10 μM ADR and 1 μM PMA were combined the effects were additive, with a maximum decrease in the Ca 2+ transient amplitude of 50%. A similar effect was seen on the Ca 2+-ATPase activities in isolated membranes. In erythrocytes labeled with [ 32P]orthophosphate 10 μM ADR induced a 1.5-fold increase in the phosphorylation of the Ca 2+ pump and when combined with 1 μM PMA the phosphorylation was greatly enhanced (2.3 times that induced by PMA alone). ADR alone and in combination with PMA was found to decrease both 32P labeling and lipid phosphate content of phosphatidylinositol 4,5-bisphosphate (PIP 2). This was accompanied by an increase in the amount of 1,2-diacylglycerol formed in response to 10 μM ADR. We conclude that low doses of ADR are able to stimulate the breakdown of 6-13% of erythrocyte PIP 2 by phospholipase C at an intracellular calcium concentration of 2.5 μM, normally regarded as below threshold for phospholipase C activation in erythrocytes. The diacylglycerol formed appears to stimulate protein kinase C to activate the Ca 2+ pump and enhance its phosphorylation and Ca 2+ efflux in intact human erythrocytes.

Phosphatidylcholine Turnover in Activated Human Neutrophils.: AGONIST-INDUCED CYTIDYLYLTRANSFERASE TRANSLOCATION IS SUBSEQUENT TO PHOSPHOLIPASE D ACTIVATION

Phosphatidylcholine synthesis and degradation are tightly regulated to assure a constant amount of the phospholipid in cellular membranes. The chemotactic peptide fMLP and the phorbol ester, phorbol 12-myristate 13-acetate, are known to stimulate phosphatidylcholine degradation by phospholipase D in human neutrophils. fMLP alone triggered phosphatidylcholine breakdown into phosphatidic acid, but did not stimulate phosphatidylcholine synthesis or activation of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase. Adding cytochalasin B to fMLP led to some conversion of phosphatidic acid into diglyceride, and fMLP was then able to trigger choline incorporation into phosphatidylcholine, and cytidylyltransferase translocation from cytosol to membranes. Inhibition of phosphatidyl-choline-phospholipase D activation with tyrphostin led to inhibition of choline incorporation. Therefore, phosphatidic acid-derived diglyceride but not phosphatidic acid alone was effective to promote cytidylyltransferase translocation. With phorbol 12-myristate 13-acetate as agonist, and by selective labeling of phosphatidylinositol and phosphatidylcholine, we demonstrated that only phosphatidylcholine-derived diglyceride participated in cytidylyltransferase translocation. Oleic acid stimulated phosphatidylcholine synthesis, but induced a weak increase in diglyceride and a slight cytidylyltransferase translocation, and did not stimulate phospholipase D activity. Our data established that only diglyceride derived from phosphatidylcholine degradation by the phospholipase D/phosphatidate phosphatase pathway are required for agonist-induced cytidylyltransferase translocation and subsequent choline incorporation into phosphatidylcholine.

Modulation of NMDA effects on agonist-stimulated phosphoinositide turnover by memantine in neonatal rat cerebral cortex.

1. The ability of memantine (1-amino-3,5-dimethyladamantane) to antagonize the modulatory effects of N-methyl-D-aspartate (NMDA) on phosphoinositide turnover stimulated by muscarinic cholinoceptor- and metabotropic glutamate receptor-agonists has been examined in neonatal rat cerebral cortex slices. 2. Memantine antagonized the inhibitory effect of NMDA (100 microM) on both total [3H]-inositol phosphate ([3H]-InsPx) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulations stimulated by carbachol (1 mM) with EC50 values of 21 and 16 microM respectively. 3. Memantine concentration-dependently antagonized (IC50 24 microM) the ability of NMDA (10 microM) to potentiate [3H]-InsPx accumulation in response to a sub-maximal concentration of the metabotropic glutamate receptor agonist, 1S,3R-ACPD (10 microM). 4. The small (approx. 3 fold), concentration-dependent increase in [3H]-InsPx accumulation stimulated by NMDA was completely antagonized by the prototypic NDMA receptor-channel blocker, MK-801 (1 microM) at all concentrations of NDMA studied (1-1000 microM). In contrast, antagonism by memantine (100 microM) was observed only at low concentrations of NMDA (1-10 microM), whilst [3H]-InsPx accumulation stimulated by high concentrations of NMDA (300-1000 microM) was markedly enhanced by memantine. 5. Assessment of the incorporation of [3H]-inositol into inositol phospholipids revealed that memantine (100 microM) caused an approximate 2 fold increase in the labelling of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. 6H.p.l.c. separation of [3H]-inositol (poly)phosphates demonstrated that whilst memantine (100 microM)alone had no significant effect on the accumulation of any isomer, it substantially altered the profile of accumulation stimulated by NMDA (1 mM), greatly facilitating accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4).7.These data provide evidence that memantine can antagonize the actions of NMDA in neonatal rat cerebral cortex slices in a manner consistent with this agent acting as a NMDA receptor-channel blocker. In addition, at least two further actions of memantine can be proposed. Memantine increases the rate of [3H]-inositol incorporation into the cellular inositol phospholipid fraction, without significantly stimulating phosphoinositide turnover. Furthermore, memantine can substantially alter patterns of inositol (poly)phosphates stimulated by NMDA, promoting the accumulation of the established and putative second messengers Ins(1,4,5)P3 and Ins(1,3,4,5)P4 which are not increased by NMDA in the absence of memantine. It is unknown whether these latter loci of memantine action contribute to known therapeutic actions of this agent.

Hydrolysis of cell surface inositol phospholipid leads to the delayed stimulation of phosphatidylinositol synthesis in bovine aortic endothelial cells

In order to address the issue of how inositol phospholipid synthesis is controlled in a resting cell we looked for enhanced [ 3H]phosphatidylinositol (PtdIns) labelling in response to the hydrolysis of cell surface PtdIns. Bacillus thuringiensis PtdIns-PLC when added to intact bovine aortic endothelial (BAE) cells rapidly hydrolysed 9.1 ± 1% of the total cellular PtdIns. This result suggests that BAE cells have a cell surface pool of PtdIns. Hydrolysis of cell surface PtdIns, in contrast to the agonist-stimulated hydrolysis of inner leaflet PtdIns, did not lead to a rapid (minutes) stimulation of PtdIns resynthesis. Prolonged incubation of BAE cells with PtdIns-PLC led to further hydrolysis of PtdIns (up to 20% of total cellular PtdIns). This second phase of PtdIns-PLC induced hydrolysis was inhibited by the addition of brefeldin A suggesting that it was dependent on vesicular traffic to the plasma membrane from the endoplasmic reticulum. Furthermore, the above result suggests that prolonged incubation of intact cells with PtdIns-PLC leads to the slow depletion of intracellular PtdIns stores. This second phase of PtdIns-PLC induced hydrolysis was associated with PtdIns resynthesis since prolonged incubation with PtdIns-PLC, but not B. cereus PtdCho-PLC (which does not hydrolyse PtdIns), led to enhanced PtdIns labelling. The results indicate that extracellular PtdIns-PLC induced PtdIns resynthesis may occur due to PtdIns-PLC induced intracellular PtdIns depletion.

Effect of different sepsis-related cytokines on lipid synthesis by isolated hepatocytes

Cytokines seem to play an important role in the metabolic disturbances that are commonly associated with sepsis. In this study, we analyzed the effect of tumor necrosis factor, interleukin-1 and interleukin-6, as well as that of tumor necrosis factor in combination with interleukin-1 or interleukin-6, both on free fatty acids and on phospholipid synthesis by isolated rat hepatocytes. All three cytokines and combinations caused inhibited D-[U-14C]glucose incorporation into phosphatidylcholine (tumor necrosis factor = 6.39 +/- 1.13 pmol/microgram protein vs. control = 12.90 +/- 0.98 pmol/microgram protein, n = 7; p < 0.001). However, when [U-14C]palmitate was used as radioactive precursor, tumor necrosis factor, either alone or in the presence of the other cytokines, stimulated phosphatidylcholine synthesis. D-[U-14C]glucose incorporation into free fatty acids and triacylglycerol was also significantly stimulated, whereas phosphatidylinositol labeling was found inhibited by the assayed cytokines. Our results demonstrate an effect of sepsis-related cytokines, more evident for tumor necrosis factor, on hepatocyte lipid synthesis either from glucose or palmitate. Also, the findings support the hypothesis that cytokine-induced changes in hepatocyte lipid synthesis can contribute to the impairment in lipidic metabolism seen in patients with sepsis.

Effects of perphenazine on the metabolism of inositol phospholipids in SK-N-BE(2) human neuroblastoma cells

Administration of myo-[ 3H]inositol to SK-N-BE(2) human neuroblastoma cells for 24 hr resulted in equilibrium labelling of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP 2), as well as in retention of a large intracellular pool of free myo-[ 3H]inositol. Equilibrium labelling was no longer observed when cells were treated for 2 hr with 20 μM perphenazine (PPZ) in label-free medium; under these conditions, myo-[ 3H]inositol from the retained intracellular pool was incorporated into PI and PIP but not into PIP 2. Analysis of water-soluble myo-[ 3H]inositol derivatives and inositol 1,4,5-trisphosphate mass determination indicated that PPZ did not stimulate phosphoinositide hydrolysis by phospholipase C. These results indicate that PPZ raises PI and PIP levels, whereas it is ineffective in expanding the PIP 2 pool. The latter effect is not due to a concomitant synthesis and hydrolysis of this lipid.

Distribution of gentamicin to the cochlea of the chicken embryo.

Aminoglycoside antibiotics are ototoxic in mammals and birds, including recently hatched chicks, but chicken embryos are insensitive to the ototoxicity of gentamicin, kanamycin, and streptomycin. To determine whether or not the insensitivity is due to a lack of antibiotic distribution to the avian cochlea, the distribution of gentamicin to the cochlea of the White Leghorn chicken embryo was compared to the distribution to the cochlea of the recently hatched White Leghorn chick. Fertile eggs were injected with a maximally tolerated dose of gentamicin sulfate (0.1 mg/egg/day) on incubation days 10-18, and the chicks were injected subcutaneously with either 5 mg (non-ototoxic) or 100 mg (ototoxic) gentamicin sulfate/kg body weight on days 1-9 after hatching. Gentamicin sulfate was histochemically detected within the basilar papilla (the avian equivalent of the organ of Corti) in all treated chicken embryos and chicks by 1 day after the first injection, and the staining was intense after 3 days of treatment. By ultrastructural immunocytochemistry, mild, diffuse labeling for gentamicin sulfate was detected within the endoplasmic reticulum of short and tall hair cells of chicken embryos by incubation day 17. Moderate labeling of gentamicin sulfate was detected in the infracuticular region of lysosomes of hair cells in chicks receiving 5 treatments of gentamicin sulfate at 5.0 mg/kg body weight and after 1 treatment of gentamicin sulfate at 100 mg/kg body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor effect of piericidin B1 N-oxide.

Piericidin B1 N-oxide was isolated from a culture broth of Streptomyces sp. as a novel inhibitor of phosphatidylinositol (PI) turnover. Piericidin B1 N-oxide specifically inhibited orthophosphate labeling of PI induced by epidermal growth factor (EGF) without affecting the formation of phosphatidic acid (PA). Like piericidins A1 and B1, piericidin B1 N-oxide inhibited ATP synthesis in A431 cells; however, the effect of piericidin B1 N-oxide on PI synthesis was stronger than that of piericidins A1 and B1. At the concentration inhibiting PI synthesis, piericidin B1 N-oxide showed no inhibitory effect on DNA, RNA, or protein synthesis. We also demonstrated that piericidin B1 N-oxide reversibly inhibited the growth of A431 cells in situ and suppressed the growth of Ehrlich carcinoma in vivo when administered to mice by intraperitoneal (ip) injection.

Elongation, desaturation, and esterification of essential fatty acids by fetal rat brain in vivo.

Tracer amounts of either [1-14C]linolenic (18:3n-3, LNA), or [1-14C]linoleic (18:2n-6, LA) acids were intracranially injected into 19- to 20-day-old rat fetuses, and the time course of the in vivo formation and esterification of their long chain polyenoic metabolites was determined for up to 20 h. A rapid disappearance of free LNA and LA, with apparent half-lives of 60 and 40 min, respectively, was noticed. One hour after LNA injection, 32.3% and 14.3% of the total brain radioactivity was found in the neutral glyceride (NG) and phospholipid (PL) fractions, respectively. After 20 h, PL radioactivity attained a level of 75%. Phosphatidylcholine (PC), diacylglycerol (DG), and triacylglycerol (TG) constituted 40%, 23%, and 9% of the total brain label at 1 h, and 35%, 10% and 14% at 20 h, respectively. Ethanolamine-containing PL (including plasmalogen) radioactivity accounted for about 10% up to 6 h and increased nearly 3-fold at 20 h, primarily due to an increase in the amount of labeled docosapentaenoic and docosahexaenoic acids (DHA), the elongation-desaturation products of LNA. A similar pattern of incorporation into NG and PL fraction was observed after the administration of [1-14C]LA. After 1 h, PC, DG, and TG species constituted 23%, 10%, and 23% of the total brain radioactivity, whereas after 20 h it accounted for 44%, 6%, and 10%, respectively. Although radioactivity in the ethanolamine PL also increased substantially from 4% at 1 h to 29% at 20 h, the main labeled fatty acid in this fraction was LA. Labeled arachidonic acid (AA) constituted 42.7% of the total radioactivity in phosphatidylinositol (PI) at 20 h. At this time, it comprised 12.5% and 14% of the total radioactivity in PC and ethanolamine PL, respectively, suggesting a high degree of esterification selectivity. Comparison of the total amounts of LA and LNA and their corresponding labeled AA and DHA metabolites in brain and liver after 3 and 6 h indicated that the contribution of liver metabolism to the elongation-desaturation under these conditions was negligible. One hour after intracerebral injection of [3H]DHA (22:6n-3) or [3H]AA (20:4n-6), 29.2% and 12% of total radioactivity, respectively, was found in the ethanolamine PL while 20% and 40% was incorporated in PC, respectively. PI labeling by [3H]AA was 6- to 8-fold higher than that seen in the presence of DHA. A high percent of radioactivity (26.9% and 18.2%) was found in DG and TG species.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the alpha 1B-adrenergic receptors of chicken hepatocytes. Signal transduction and actions.

1. In chicken hepatocytes, alpha 1-adrenoceptor activation increased: (a) phosphatidylinositol labeling; (b) production of inositol trisphosphate; (c) cytosol calcium; and (d) phosphorylase activity. 2. Prazosin (Ki approximately 0.2-0.4 nM) was more potent in inhibiting these actions than 5-methyl-urapidil (Ki approximately 30-60 nM); these actions were sensitive to chlorethylclonidine suggesting the involvement of alpha 1B-adrenoceptors. 3. The stimulation of phosphoinositide turnover was insensitive to pertussis toxin. 4. In chicken liver membranes, [3H]prazosin binding sites (Bmax 872 fmol/mg protein) with high affinity for prazosin (KD 0.3 nM; Ki 0.4 nM) and lower affinity for 5-methyl-urapidil (Ki 46 nM) were detected, consistent with the presence of alpha 1B-adrenoceptors.

Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.

Pharmacological evidence for interactions between 5-HT1A receptor agonists and subtypes of α1-adrenoceptors on rabbit aorta

This study was designed to determine if α1-adrenoceptors are involved in the vascular responses to 5-HT1A receptor agonists. Buspirone (3.1 × 10−7–3.1 × 10−5 M) and 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT; 3.1 × 10−6–10−4 M) el elicited contractions of rabbit aorta rings which were blocked by prazosin (10−9–5.6 × 10−9 M), but which were unaffected by reserpine pretreatment (1 mg/kg i.p.). 5-Methylurapidil (10−7 and 10−6 M) blocked contractions elicited by 8-OH-DPAT and by buspirone, whereas chloroethylclonidine (10−5 and 10−4 M) inhibited only the effect of buspirone. In addition, these 5-HT1A receptor agonists relaxed arteries precontracted with α-adrenoceptor agonists in a similar range of concentrations in which they elicited contraction. Moreover, 8-OH-DPAT and buspirone protected the α-adrenoceptors from the irreversible blockade provoked by phenoxybenzamine (10−7 M), as judged by the norepinephrine contraction and stimulated phosphatidylinositol labeling. According to these results the contractile and relaxant effects elicited by 5-HT1A receptor agonists are a consequence of a direct interaction with α1-adrenoceptors. The contraction elicited by 8-OH-DPAT may be mediated by α1A-adrenoceptors, whereas both α1A- and α1B-adrenoceptors may mediate the effect of buspirone in rabbit aorta.

Guinea pig hepatocyte α 1A-adrenoceptors: characterization, signal transduction and regulation

Activation of guinea pig hepatocyte α 1-adrenoceptors increases phosphatidylinositol (PI) labeling, [ 3H]inositol phosphate production and phosphorylasc activity. These adrenergic actions were not altered by pretreatment with chlorethylclonidine but were blocked by 5-methyl urapidil and prazosin (the former being 3- to 10-fold more potent than the latter), indicating that α 1A-adrenoceptors were involved. When the cells were incubated in buffer without calcium and containing EGTA, the α 1A-adrenergic stimulation of PI labeling was diminished but not abolished and that of phosphorylasc was not affected. The α 1A-adrenergic effects were insensitive to pertussis toxin treatment. Phorbol myristate acetate inhibited the α 1A-adrenergic actions, although at relatively large concentrations, and also those of other agents such as angiotensin II and NaF. Our data clearly indicate that guinea pig hepatocytes express α 1A-adrenoceptors whose activation stimulates phosphoinosilide turnover, via a pertussis toxin-insensitive process; the α 1A-adrenergic effects were at least partially independent of extracellular calcium.

Insulin and progesterone increase 32PO 4-labeling of phospholipids and inositol 1,4,5-trisphosphate mass in Xenopus oocytes

After a 4–6 h induction period, insulin or progesterone induces Xenopus oocytes to enter prophase of meiosis. During the period of induction, both insulin and progesterone induced an increase in 32PO 4 labeling of phosphatidylcholine and phosphatidylinositol. Through a mass assay, we found that insulin and progesterone increase inositol 1,4,5-trisphosphate (IP 3) at about 15–30 s, 15 min and at about 2–3 h (0.5 GVBD 50) after hormone addition. Since IP 3 increases were small (from a basal of 66 to 104 nM), the results agree with prior conclusions that progesterone does not induce a large, cytosolic calcium elevation. Insulin is probably acting through the insulin-like growth factor-1 receptor as insulin concentrations greater than about 50 nM are required to increase IP 3.

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [(32)P]phosphate and [(3)H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10(9) platelets) in amount of extractable PIP(2) when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP(2) (1.09±0.11 nmol/10(9) platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [(3)H]inositol label in PIP(2) in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP(2) and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the (3)H-labelled material with interfacial protein. Blocking thromboxane A(2) formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and (32)P-labelling of phosphatidic acid and (3)H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [(3)H]inositol of the phosphoinositides in unstimulated platelets differed (PIP(2)> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP(2), indicating that the increase in PIP was due to degradation of PIP(2). Thus, the large decrease in extractable PIP(2) and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP(2) to PIP.